Cytokine-neuroantigen (NAg) fusion proteins represent a novel therapeutic platform for the treatment of multiple sclerosis. The purpose of this proposal is to address mechanisms by which these fusion proteins act as tolerogenic, therapeutic vaccines (TTV) in murine models of experimental autoimmune encephalomyelitis (EAE). Previous research showed that homologous fusion proteins were effective NAg-specific TTV in rat models of EAE. The fusion proteins used in the proposed research will contain murine interferon (IFN)-beta or granulocyte-macrophage colony-stimulating factor (GM-CSF) as immunoregulatory cytokines. The relevant NAg will include MOG35-55 and PLP139-151 for the C57BL/6 (B6) and SJL mouse models of EAE, respectively. The hypothesis is that TTV function by cytokine-mediated conditioning of APC and the consequent targeting of tethered NAg to those conditioned APC. Specific Aim 1 will focus on the structural requirements of TTV in murine EAE. Mechanisms of cytokine-mediated antigen targeting of APC will be assessed by the requirement for covalent cytokine-NAg linkage for tolerance induction in vivo and antigenic potentiation in vitro. These TTV will be tested as a pre-treatment in a tolerance induction regimen and separately as an intervention after EAE onset. The experimental design will consist of five treatment groups;(a) saline, (b) peptide alone, (c) cytokine alone, (d) a mixture of cytokine and NAg as independent molecules, and (e) a cytokine-NAg TTV. The prediction is that a covalently linked cytokine-NAg TTV will be required for disease inhibition in vivo and potentiated antigen recognition in vitro. Specific Aim 2 will focus on mechanisms of TTV action in EAE. Two subaims will address mechanisms by which GMCSF-based and IFN-beta-based TTVs regulate EAE in B6 mice. These aims will assess whether TTV inhibit encephalitogenic Th1 and Th17 effector T cells and whether the cytokine domains of TTV require specific interactions with the respective cytokine receptors in vivo to elicit tolerance. Overall, the project is designed advance the development of a TTV as a neuroantigen-specific intervention to control and reverse multiple sclerosis, a devastating disease that afflicts nearly 350,000 Americans and over 2 million persons in the western world. PUBLIC HEALTH RELEVANCE: The project will advance the development of a tolerogenic vaccine as a neuroantigen-specific intervention to control and reverse multiple sclerosis, a devastating disease that afflicts nearly 350,000 Americans and over 2 million persons in the western world. A neuroantigen-specific tolerogenic vaccine will have qualitative advantages over current therapy, particularly in that a limited set of vaccinations should have enduring clinical benefit thereby preempting the need for chronic drug administration. The current project is a critical step in advancing this protein-based therapeutic from the bench through a comprehensive clinical development path in preparation for an Investigational New Drug (IND) application to the Food and Drug Administration (FDA).